Binding of 3,4,5,6-tetrahydroxyazepanes to the acid-β-glucosidase active site: implications for pharmacological chaperone design for Gaucher disease - PubMed (original) (raw)

. 2011 Dec 13;50(49):10647-57.

doi: 10.1021/bi201619z. Epub 2011 Nov 14.

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Binding of 3,4,5,6-tetrahydroxyazepanes to the acid-β-glucosidase active site: implications for pharmacological chaperone design for Gaucher disease

Susan D Orwig et al. Biochemistry. 2011.

Abstract

Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-β-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

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Figures

Fig. 1

Fig. 1

Chemical structure of the natural GCase substrate, GlcCer, representative azasugars investigated as pharmacologic chaperones, IFG, NB- and NN- DNJs, as well as the azepane compounds 1, 2, and 3 described in this study.

Fig. 2

Fig. 2

Competitive inhibition curves for 1, 2, and 3, respectively, toward GCase. Inset: IC50 values. Error bars indicate standard deviation.

Fig. 3

Fig. 3

Ball-and-stick representation of the GCase active site upon compound binding. (A) 2 (B) 1 (C) NN-DNJ (PDB code 2V3E) (D) IFG (PDB code 2NSX). Difference (Fo— Fc) electron density for 1 and 2 was calculated from the initial phasing solution using only protein coordinates and is contoured to 3σ. Hydrogen bonding interactions are indicated by dashed black lines and represent distances between 2.5 Å– 3.5 Å.

Fig. 4

Fig. 4

Superposition of 1 and 2 bound GCase structures and comparison of loops adjacent to the active site (inset). After binding, Loop 1 adopts either a helical turn as seen for compound 1 (inset, yellow) and IFG (inset, green), or an extended loop conformation seen in the compound 2 (inset, orange) and glycerol (inset, blue) bound structures. Changes in Loop 2 are due to crystal packing (see text).

Fig. 5

Fig. 5

Comparison of Loop 1 configuration. (A) IFG-, (B) 1-, and (C) 2- bound GCase. Top: orientation of Tyr 313 relative to Glu 340. Bottom: interactions of loop with interior GCase helix harboring Asn 370. Hydrogen bonding interactions are indicated by dashed black lines.

Fig. 6

Fig. 6

Effects of 1, 2, and 3 on mutant GCase activity in intact patient derived fibroblasts G202R (A) and N370S/V394L (B). Enzyme activity is normalized to untreated and assigned a relative activity of 1. The right-hand axis is the residual activity of the mutant expressed as the percentage of WT GCase activity. Mean values for triplicate experiments are shown. # = p < 0.05.

Scheme 1

Scheme 1

Synthesis of Di-epoxide S-3

Scheme 2

Scheme 2

Synthesis of Tetrahydroxyazepane 1

Scheme 3

Scheme 3

Synthesis of Tetrahydroxyazepanes 2 and 3

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